固氮酶铬铁蛋白和锰铁蛋白大单晶的培养

用液/液扩散法, 从分别含Cr和Mn的无氨培养基中生长的固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化出的CrFe蛋白和MnFe蛋白, 在合适的结晶条件下生长出深棕色大单晶(最大晶体的尺寸分别为0.20 mm×0.20 mm×0.07 mm 和 0.18 mm×0.18 mm×0.05 mm).PEG、MgCl2和NaCl 的浓度对这两种蛋白的出晶时间、晶核数目、晶体大小和形状都有明显影响.结果表明,用此结晶法有利于CrFe蛋白和MnFe蛋白生长出可供X-射线衍射分析的大单晶.     

T-DNA插入水稻群体中卷叶突变体R1-A2的遗传分析

在根癌农杆菌介导的T-DNA(携带有除草剂Basta抗性基因bar和Ds因子)转化中花11水稻群体中,获得了一个叶片发生明显内卷的突变体R1-A。经过连续三代的分离鉴定,获得突变体的纯合株(R1-A2),并与中花11号进行杂交,在调查的36个F_1植株中,全部表现为卷叶,并对Basta除草剂都表现为抗性。在852个F_2单株中,卷叶为645株,正常叶207株,卷叶和正常叶的比例为3:1,其中,卷叶株均对Basta表现抗性,正常叶株均对Basta表现敏感,表明卷叶性状和Basta抗性存在着共分离关系。用扩增Ds因子的引物,对F_2中45个卷叶抗性株进行PCR鉴定,都获得预期长度的Ds因子片段,进一步表明在这些卷叶的植株中都有T-DNA的插入;而30个正常叶敏感株都不能检测到Ds的特征片段。在以卷叶突变(R1-A2)为回交亲本的F_1B_1植株中,全部植株表现卷叶;在以中花11号为回交亲本的F_1B_1植株中,卷叶和正常叶植株的分离比为1:1。上述结果表明该卷叶突变是个显性突变,受一个基因所控制,且该基因的突变与T-DNA的插入有关。     

尿道致病性大肠杆菌U17株rstA缺失株降低对小鼠的致病性

【目的】探究双组份系统RstA/RstB中效应基因rstA对尿道致病性大肠杆菌毒力的影响。【方法】利用λRed重组系统构建UPEC U17 rstA缺失株U17ΔrstA,并构建相应的回复株,通过体内外试验评价rstA基因缺失对UPEC毒力的影响。【结果】生长曲线的测定结果显示,在LB普通培养基中,缺失株生长速度与野生株相似,但在LB贫铁培养基中,缺失株生长速度较野生株相比明显下降;体外环境应激试验结果显示,缺失株在强酸、强碱、高渗透压、尿素、氧化应激等环境压力下的生存能力与野生株相似;菌株生物被膜检测结果显示,缺失株的生物被膜形成能力与野生株相当;荧光定量PCR检测结果显示,rstA基因在贫铁环境下的表达水平较正常条件下显著上调,暗示贫铁环境可能是rstA发挥效应的刺激信号。6周龄BALB/c小鼠尿道感染试验结果显示,rstA缺失株在尿液、膀胱、肾脏中的带菌量显著低于野生株,而回复株毒力恢复至野生株水平,表明rstA缺失能显著降低UPECU17的毒力。【结论】rstA与尿道致病性大肠杆菌的致病性相关,为潜在的毒力因子。     

含锰固氮酶组分Ⅰ蛋白的纯化和特性

棕色固氮菌（Azotobacter vinelandii Lipmann)突变种UW3能在无Mo的无氨培养基中固氮生长,低浓度的Mn对UW3突变种生长有促进作用,从在Mn中生长的UW3菌体中分离得到的部分纯固氮酶组分Ⅰ蛋白含量有Mn和Fe原子（Fe/Mo/Mn为10．41：0．19：1．00）并有OP MoFe蛋白一半的还原乙炔和质子的活性。这种蛋白的吸收光谱和圆二色谱与MoFe蛋白存在明显的差异,含Mn蛋白的亚基分子量都与MoFe蛋白的α亚基相近。初步结果表明,这种含Mn蛋白可胡是一种固氮酶组分Ⅰ蛋白。     

产小檗碱诱变菌株培养条件的优化

S-NU-3-2菌株是对源自药用植物黄檗的内生真菌S6进行诱变获得的小檗碱产量比出发菌株有明显提高的突变株,本研究对其培养条件进行了优化,以期进一步提高其产量,为开发利用真菌发酵生产植物活性成分的新途径奠定基础。以菌丝生长和小檗碱产量为指标,筛选出了适宜该菌株发酵的基本培养基、碳源、氮源、光照和培养温度,并经进一步的正交试验优化,得出该高产菌株的最佳培养条件为豆芽汁基本培养基含蔗糖3%,酵母膏0.2%,pH7.0,温度26℃,全光照培养。与初始培养条件相比,在优化后的条件下,该菌株的小檗碱得率提高了47.2%,菌体生物量提高了24%。     

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.     

What limits meiotic crossovers?

Comment on: Crismani W, et al. Science 2012; 336:1588-90.     

Cometabolic turnover of aniline,phenol and some of their monochlorinated derivatives by the Rhodococcus mutant strain AM 144

One-step conversion of aniline, phenol and some of their monochlorinated derivatives into the corresponding catechols by resting pre-adapted cells of the Rhodococcus mutant strain AM 144 (defective in synthesis of catechol 1,2-dioxygenase) was shown to depend on the availability of an additional metabolizable carbon substrate, e.g. glucose or acetate. A stoichiometric relation existed between the amount of the latter compounds added and the amount of aniline (or phenol, respectively) converted into catechol suggesting that the primary function of the cosubstrates was to provide reducing power to the oxygenative transformation reaction. The observed cosubstrate-dependence generally parallels that seen in previous studies on turnover of different monochloroaromatic non-growth substrates by aromatics-utilizing Rhodococcus wildtype-strains. Cell cultures of strain AM 144 growing at the expense of acetate also proved able to convert aniline into catechol. Typically, growth of the cells was retarded during the phase of aniline transformation as compared to the respective control cultures. Based on the results of these model experiments, it was concluded that (i) in natural microbial communities cometabolically active bacteria would hardly enrich under cometabolic conditions over fast-growing non-cometabolizing bacteria if the latter organisms will tolerate the particular non-growth substrate, and (ii) cometabolizing bacteria would have a selective advantage only if the non-growth substrate to be transformed is a toxic one or if it can serve as a potential nutrient source (e.g., of nitrogen or sulfur).Abbreviations MCA monochloroaniline - MCP monochlorophenol - MCC monochlorocatechol - TLC thin-layer chromatography - MS mass spectrometry - GLC gas-liquid chromatography - UV ultraviolet (range of the spectrum)     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).